1. Field of the Invention
The invention relates to the field of cell biology. In detail it relates to the obtainment of mesenchymal stem cells from human tissue. This invention might be applicable in healing within the frame of the treatment of several diseases.
2. Related Prior Art
Currently in biomedical sciences a new field is developing: cell therapy. By means of cell transplantation insufficient activities of tissue functions can be redressed and affected organs can be regenerated. The function of renewal and restoration is taken over from stem cells in vivo which form an agglomeration of non-differentiated precursors of different cells types which are kept in reserve. As a result of this the use of stem cells is a promising field of cell therapy. In this connection of particular importance is the obtainment of stem cells from human tissue.
At present one has been succeeded in obtaining different types of stem cells from the adult human organism. In detail they concern hematopoietic (blood cell precursors), neuronal (precursors of nerve tissue cells), mesenchymal cells (cells which are capable to differentiate into cells of mesenchymal origin as well as into other embryonic layer cells) and other.
It is characteristic for mesenchymal stem cells that they can be obtained and cultivated comparatively easily; they are also capable to proliferate in vitro over prolonged periods of time. Furthermore, they excel in a broad spectrum of differentiation. In this connection the attention is especially directed to the obtainment of mesenchymal stem cells from the adult human organism. However, till this day there is no universal method for the obtainment of stem cells.
For the first time stern cells could be obtained from the spinal marrow due to their ability of adhering to the surface of culture dishes (Fridenshtein A. J., Deriglazova U. F., Kulagina N. N. et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp. Hematol. 1974 Vol. 2. P. 83-92).
The disadvantage of this method consists in the lacking homogeneity of the obtained cell population. Nevertheless the adhesion of mesenchymal stem cells to plastics has been established as starting basis for further improved methods for obtaining mesenchymal stem cells.
A method for obtaining mesenchymal stem cells is known that results from the cells' ability of adhering to the surface of culture dishes, whereby specific batches of the embryonic bovine serum are used. Thus it was managed to obtain cells with a high ability of adhesion, high rate of proliferation and long maintaining multipotency (Heynesworth S. E., Goshima J., Goldberg V. M., Calplan A. I. Characterization of cells with osteogenic potential from the human bone marrow//Bone. 1995. Vol. 13. P. 81-85.)
The disadvantage of this method consists in the fact that the analysis of the serum during the search for a batch that is suitable for the cultivation of cells plenty of time and work is required; furthermore the reproduction of results is not possible.
It is known to obtain mesenchymal stem cells by a method by means of which a selection for antibodies against Stro-1 (antigen with unknown function) is performed, Stro-1 is temporarily expressed on the surface of mesenchymal stem cells (Grontos S., Simmons P. J. The growth factors requirements of Stro-1 positive human stromal precursors under serum-deprived conditions in vitro//Blood. 1995. Vol. 85. P. 929-940).
This method concerns an extreme complex process that is very time consuming due to the preliminary antibody preparation.
A method for obtaining mesenchymal stem cells is known by means of which mononuclear cells are obtained by centrifugation in a Ficoll gradient, a selection for antibodies against the surface antigen CD105 which is expressed on the surface of mesenchymal stem cells, as well as by a cultivation of cells which adhere to plastics. The portion of CD105+ cells amount to 2 to 3% of the mononuclear cells. As a result it was possible to obtain a cell population which shows the morphology and the expression profile of the surface antigens which are characteristic for mesenchymal stem cells and which also have chondregenic potential (Majumdar M. K., Banks V., Peluso D. P., Morris E. A. Isolation, characterization, and chondregenic potential of human bone marrow-derived multipotential stromal cells//J. Cell. Physiol. 2000. Vol. 185. P. 98-106).
This method enables the obtainment of the CD105+ cell fraction which is enriched in mesenchymal stem cells; however, this method requires an additional selection stage in which antibodies immobilized to magnetic beads are used. Thereby a portion of cells gets lost so that additional efforts are required.
It is known that with increasing age the number of mesenchymal stem cells in the human organism is diminished conspicuously. This also apply to their ability to proliferate and differentiate (Rao M. S., Mattson M. P. Stem cell and aging: expanding the possibilities//Mech. Ageing Dev. 2001. Vol. 122. P. 713-734). For this reason one is working on the preparation of mesenchymal stem cells with high proliferative activity and potential for differentiation, which are for example originating from fetal tissue or larvae organs, respectively.
A method for obtaining mesenchymal stem cells from fetal progeny blood is known in the art. To obtain them the mononuclear cell fraction is prepared by centrifugation in the Ficoll gradient and they are cultivated under conditions which are beneficial for the growth of mesenchymal stem cells. The so obtained cells show osteogenetic or adipogenetic potential, respectively (Campagnoli C., Roberts I. A., Kumar S. et al. Identification of mesenchymal stem/progenitor cells in human first trimester fetal blood, liver, and bone marrow//Blood. 2001. Vol. 98. P. 2396-2402).
The disadvantage of the obtainment of mesenchymal stem cells from this source consists in the fact that fetal tissue is problematic in view of its accessibility. Moreover one has to face ethic problems which are associated with its use. Furthermore, it has turned out that the obtained cell populations are not homogeneous: 76% of the samples contained osteoclast-like cells; they expressed the characteristic antigens CD45, CD51/CD61, and were negative for CD64 (marker for macrophages), SH2 (marker for mesenchymal stem cells), CD31 (marker for endothelial cells). Only 26% of the samples were composed of cells which were similar to mesenchymal stem cells and expressed SH2, SH3, SH4, MAB1470, CD13, CD29, CD49e, CD54, CD90, ASMA, and were negative for CD31 and vWF (marker for endothelial cells).
Known is the method for obtaining mesenchymal stem cells from the subendothelial layer of the umbilical vein (Romanov Y. A., Svinitskaya V. A., Smirnov V. N. Searching for alternative sources of postnatal human mesenchymal stem cells; candiate MSC-like cells from umbilical cord//Stem Cells. 2003. Vol. 21. P. 105-110). For these purposes the umbilical vein was treated inside with a collagenase IV solution for a short time (for 15 minutes). The so obtained cells were cultivated in DMEM-LG supplemented with 10% FBS. In this manner a cell population with fibroblast-like morphology was obtained; it expressed a range of antigens which was similar to mesenchymal stem cells: ICAM1+/−, VCAM1+, CD34−; MySM− (smooth muscle myosine); CD31−, vWF− (marker for endothelial cells); CD14−, CD45−, CD68− (marker for monocytes/macrophages), however it did express smooth muscle fibre actin ASMA. The so obtained cell population showed osteogenetic or adipogenetic potential in vitro.
The disadvantage of this method consists in the heterogeneity of the obtained cell population: The primary culture contained endothelial and smooth muscle cells, whereby the endothelial cells have not proliferated under these conditions, whereas the myocytes portion was maintained during the cultivation.
A method for obtaining mesenchymal stem cells from human lipoaspirate is known in the art. In this method fat tissue reduced to small pieces is exposed to the effect of collagenase (type I). After neutralization of the collagenase and rinsing of the cell suspension a purification is performed in order to remove cell residues by the use of filters having a pore size of 100 μm. By doing so a population of mesenchymal stem cells could be obtained that showed a characteristic morphology, immunophenotype and that was able to differentiate into bone, cartilage, fat, muscle or nerve tissues, respectively (Zuk P. A., Zhu M., Ashjian P., De Ugarte D. D., Huang J. I., Mizuno H., Alfonso Z. C., Fraiser J. K. Benhaim P. and Hedrick M. H. Human adipose tissue is source of multipotent stem cells//Molecular Biology of the Cell. 2002. Vol. 13. P. 4279-4295).
This method has the disadvantage that the obtained cell suspension is heterogenous and the yield is low.